3D High Throughput Model to Predict Drug Efficacy in Fibrosis Progression vs Reversal

摘要：预测纤维化进展与复发中药物疗效的3D高吞吐量模型 特发性肺纤维化（IPF）是最无情的进行性和致命的纤维化肺病， 不成比例地影响老年人。尽管最近有两种药物获得了FDA批准用于IPF，但这些药物 药物仅适度减缓肺衰退的进展，并且不能改善患者的生活质量。 没有可用的疗法可以“逆转”纤维化。尽管许多团体努力开发 IPF治疗的进展非常缓慢。这一建议侧重于两个可能的原因， 这些困难：（1）目前的临床前筛选模型不能可靠地预测药物的成功， （2）虽然IPF被广泛认为是一种年龄相关疾病，但药物治疗 没有针对年龄相关的病理机制。 病理性纤维化是一种“纤维增生”过程的现有范式并未导致有效的IPF 治疗。该提案整合了成纤维细胞老化方面的专业知识和正在开发的新型IPF治疗药物 （Hecker实验室），采用尖端技术进行微生物打印和3D细胞测定（高山实验室）， 开发高通量表型细胞筛选试验以确定纤维化逆转的功效。的 建议的研究将利用正常的“对照”、老化的“衰老”和IPF人肺成纤维细胞，在小的 这些数字用于生物工程高通量表型测定，该测定将在21天内评估纤维化。 这些细胞的水性两相系统（ATPS）生物打印将用于产生微尺度收缩。 与常规测定相比，其体积小几个数量级。重要的是， 该项目将在已经收缩的载有细胞的凝胶周围反复微型打印新鲜胶原蛋白， 反复宫缩超过21天。该模型将使第一个高通量表型 具有确定候选药物对纤维化进展的功效的能力的筛选测定， 逆转新的细胞分析将被验证其识别纤维化逆转药物的能力， “Noxindoline”是一种高选择性的Nox 4抑制剂，目前正由Hecker实验室进行临床前开发。 Hecker实验室通过研究Nox 4的年龄依赖性改变， 导致持续的氧化还原失衡，并促进衰老和抗衰老， 肌成纤维细胞该提案假设，目前的治疗（Nursing和吡非尼酮）将抑制 促纤维化表型的进展（但不逆转），而用Noxindoline治疗将促进 已建立的促纤维化表型的逆转。其目标是： 目的1：开发使用非衰老细胞的纤维化进展的高通量生物打印细胞测定 目的2：监测衰老细胞和IPF患者细胞的纤维化进展和逆转

ABSTRACT: 3D High Throughput Model to Predict Drug Efficacy in Fibrosis Progression vs Reversal Idiopathic pulmonary fibrosis (IPF) is the most relentlessly progressive and fatal fibrotic lung disorder, which disproportionately affects the elderly. Although two drugs have recently gained FDA-approval for IPF, these drugs only moderately slow the progression of lung decline and do not improve quality of life for patients. There are no available therapies that can `reverse' fibrosis. Despite efforts by numerous groups to develop IPF treatments, progress has been aggravatingly slow. This proposal focuses on two possible reasons for these difficulties: (1) Current pre-clinical screening models fail to reliably predict the success of drug candidates in humans, and (2) Although IPF is widely regarded as an age-related disease, drug treatments have not targeted age-associated pathologic mechanisms. The existing paradigm, that pathologic fibrosis is a “fibro-proliferative” process, has not led to effective IPF treatments. This proposal integrates expertise in fibroblast aging and novel IPF therapeutics in development (Hecker lab) with cutting edge technologies for microscale bioprinting and 3D cell assays (Takayama lab) to develop a high throughput phenotypic cellular screening assay to determine efficacy for fibrosis reversal. The proposed studies will utilize normal “control”, aged “senescent”, and IPF human lung fibroblasts in small numbers to bioengineer a high-throughput phenotypic assay that will evaluate fibrosis over a 21 day period. An aqueous two phase system (ATPS) bioprinting of these cells will be used to create microscale contraction assays that are several order of magnitude smaller in volume compared to conventional assays. Importantly, the project will repeatedly micro-print fresh collagen around already contracted cell-laden gels to enable repeated contractions over 21 days. The proposed model will enable the first high-throughput phenotypic screening assay with the capability to determine a drug candidate's efficacy for fibrosis progression and reversal. The new cellular assay will be validated for its ability to identify fibrosis reversal drugs using “Noxindoline” a highly selective Nox4 inhibitor that is currently in preclinical development by the Hecker lab. Noxindoline was identified by the Hecker lab through studies of age-dependent alterations in Nox4 that results in a sustained redox imbalance, and promotes senescence and apoptosis-resistance of myofibroblasts. The proposal hypothesizes that current therapies (Nintedanib and Pirfenidone) will inhibit the progression of pro-fibrotic phenotypes (but not reversal), whereas treatment with Noxindoline will promote the reversal of established pro-fibrotic phenotypes. The aims are: Aim1: Develop high throughput bioprinted cellular assay for fibrosis progression using non-senescent cells Aim2: Monitor fibrosis progression and reversal of senescent cells and IPF patient cells